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PubMed2005

Genetic structure in Orchesella cincta (Collembola): strong subdivision of European populations inferred from mtDNA and AFLP markers.

Population genetic structure is determined both by current processes and historical events. Current processes include gene flow, which is largely influenced by the migration capacity of a species. Historical events are, for example, glaciation periods, which have had a major impact on the distribution of many species. Species with a low capacity or tendency to move about or disperse often exhibit clear spatial genetic structures, whereas mobile species mostly show less spatial genetic differentiation. In this paper we report on the genetic structure of a small, wingless arthropod species (Orchesella cincta: Collembola) in Europe. For this purpose we used mtDNA COII sequences and AFLP markers. We show that large genetic differences exist between populations of O. cincta, as expected from O. cincta's winglessness and sedentary lifestyle. Despite the fact that most variability was observed within populations (59%), a highly significant amount of AFLP variation (25%) was observed between populations from northwestern Europe, central Europe and Italy. This suggests that gene flow among regions is extremely low, which is additionally supported by the lack of shared mtDNA alleles between regions. Based on the genetic variation and sequence differences observed we conclude that the subdivision occurred long before the last glaciation periods. Although the populations still interbreed in the lab, we assume that in the long term the genetic isolation of these regions may lead to speciation processes.

Analysis of Variance↗
PubMed2006

Clinical implications of ZAP-70 expressionin chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a disease with an extremely heterogeneous prognosis. Because of this and because of the lack of a truly effective therapy, treatment of CLL must be adapted to the individual risk of each patient. Over the last quarter of century, prognosis of patients with CLL has been based on clinical features. In the last few years a number of biological prognostic parameters have been identified. Among them, the expression of ZAP-70 in neoplastic B lymphocytes is increasingly being recognized as of paramount importance to predict outcome. Patients with a low percentage (<20%) of ZAP-70 positive neoplastic B lymphocytes have a much better prognosis that those with a higher proportion (>20%) of ZAP-70 positive neoplastic B lymphocytes.

Biomarkers, Tumor↗
PubMed1999

Analysis of genetic variability in Anopheles arabiensis and Anopheles gambiae using microsatellite loci.

We analysed genetic variability in Anopheles arabiensis and Anopheles gambiae populations using microsatellite loci to determine whether the Rift Valley restricts the flow of genes. Deviations from Hardy-Weinberg expectations were significant, and were most likely to be due to the high frequency of null alleles observed. An. arabiensis populations occurring between 40 and 700 km apart across the Eastern arm of the Rift Valley were not differentiated (pair-wise F(ST) range: 0.0033-0.0265, P > 0.05). Neither were An. gambiae populations from Asembo Bay and Ghana (F(ST): 0.0063, P > 0.05) despite a geographical separation of about 5000 km. In contrast, significant differentiation was observed between An. gambiae populations from Asembo Bay and Kilifi (about 700 km apart; F(ST) = 0.1249, P < 0.01), suggesting the presence of a barrier to gene flow.

Alleles↗
PubMed2003

Mate choice theory and the mode of selection in sexual populations.

Indirect new data imply that mate and/or gamete choice are major selective forces driving genetic change in sexual populations. The system dictates nonrandom mating, an evolutionary process requiring both revised genetic theory and new data on heritability of characters underlying Darwinian fitness. Successfully reproducing individuals represent rare selections from among vigorous, competing survivors of preadult natural selection. Nonrandom mating has correlated demographic effects: reduced effective population size, inbreeding, low gene flow, and emphasis on deme structure. Characters involved in choice behavior at reproduction appear based on quantitative trait loci. This variability serves selection for fitness within the population, having only an incidental relationship to the origin of genetically based reproductive isolation between populations. The claim that extensive hybridization experiments with Drosophila indicate that selection favors a gradual progression of "isolating mechanisms" is flawed, because intra-group random mating is assumed. Over deep time, local sexual populations are strong, independent genetic systems that use rich fields of variable polygenic components of fitness. The sexual reproduction system thus particularizes, in small subspecific populations, the genetic basis of the grand adaptive sweep of selective evolutionary change, much as Darwin proposed.

Animals↗
PubMed2006

ZAP-70 mRNA quantification in B-cell chronic lymphocytic leukaemia.

OBJECTIVE: The mutational status of the immunoglobulin (Ig) V(H) gene in B-cell chronic lymphocytic leukaemia (B-CLL) identifies two subgroups of patients with significantly different outcomes. We investigated the association of ZAP-70 expression with IgVH mutational status in B-CLL by quantifying ZAP-70 mRNA, to evaluate its use as a surrogate marker for mutational status. The aim of this study was to develop a quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR) assay for the detection of ZAP-70 expression in a group of patients whose mutational status and cytogenetics had been determined previously. METHODS: RQ-PCR was used to analyse ZAP-70 expression from 42 B-CLL patients. B cells were purified using CD19 magnetic bead system and total RNA was isolated. RQ-PCR was performed using Taqman PCR. RESULTS: Twenty-five patients (60%) had mutated and 17 (40%) had unmutated IgVH genes; 94% (16/17) of patients with unmutated IgVH gene were ZAP-70 positive as assessed by RQ-PCR and 92% (23/25) of patients with mutated IgVH gene were ZAP-70 negative. In three patients, ZAP-70 expression and IgVH mutational status were discordant. CONCLUSION: This paper describes an RQ-PCR assay for the detection of ZAP-70 expression and confirms that IgV(H) unmutated CLL cells have a high expression of ZAP-70 in comparison with IgVH mutated CLL. This robust method acts as a surrogate marker for IgVH mutational status albeit with <100% concordance. However, it does provide better concordance with mutational status than that reported using flow cytometry.

Adult↗
PubMed1998

Microsatellite markers for genetic population studies in Glossina palpalis gambiensis (Diptera: Glossinidae).

Little is known about intraspecific variability in tsetse flies and its consequences for vectorial capacity. Microsatellite markers have been developed for Glossina palpalis gambiensis. Three loci have been identified and showed size polymorphisms for insectarium samples. G. palpalis gambiensis from Burkina Faso were also subjected to PCR to investigate then genetic variability. Amplifications were observed in different species belonging to the palpalis group. These molecular markers will be useful to estimate gene flow within G. palpalis gambiensis populations and analysis could be extended to related species.

Animals↗
PubMed1996

Monoclonal immunoglobulin A derived from peritoneal B cells is encoded by both germ line and somatically mutated VH genes and is reactive with commensal bacteria.

We transferred peritoneal cells from BALB/c mice into C.B17 scid/scid mice. Six to eight months after injection, only cells with the B1 phenotype were retained in the spleens and peritoneal cavities of these mice. The lamina propria of the intestine contained many peritoneal, donor-derived, immunoglobulin A (IgA)-producing cells. The mesenteric lymph nodes of these mice were found to be a major site of proliferation and generation of IgA plasmablasts. We established eight IgA-producing hybridomas from the mesenteric lymph nodes of such mice, and all the hybridomas reacted with different but partially overlapping fecal bacterial populations. Cloning and sequencing of the VH genes of these hybridomas showed that two hybridomas utilized germ line-encoded VH genes while the VH genes of the six hybridomas showed somatic mutations, some of which are indicative of an antigen-driven selection process.

Animals↗
PubMed2005

Induction of rapid detachment in Shewanella oneidensis MR-1 biofilms.

Active detachment of cells from microbial biofilms is a critical yet poorly understood step in biofilm development. We discovered that detachment of cells from biofilms of Shewanella oneidensis MR-1 can be induced by arresting the medium flow in a hydrodynamic biofilm system. Induction of detachment was rapid, and substantial biofilm dispersal started as soon as 5 min after the stop of flow. We developed a confocal laser scanning microscopy-based assay to quantify detachment. The extent of biomass loss was found to be dependent on the time interval of flow stop and on the thickness of the biofilm. Up to 80% of the biomass of 16-h-old biofilms could be induced to detach. High-resolution microscopy studies revealed that detachment was associated with an overall loosening of the biofilm structure and a release of individual cells or small cell clusters. Swimming motility was not required for detachment. Although the loosening of cells from the biofilm structure was observed evenly throughout thin biofilms, the most pronounced detachment in thicker biofilms occurred in regions exposed to the flow of medium, suggesting a metabolic control of detachability. Deconvolution of the factors associated with the stop of medium flow revealed that a sudden decrease in oxygen tension is the predominant trigger for initiating detachment of individual cells. In contrast, carbon limitation did not trigger any substantial detachment, suggesting a physiological link between oxygen sensing or metabolism and detachment. In-frame deletions were introduced into genes encoding the known and putative global transcriptional regulators ArcA, CRP, and EtrA (FNR), which respond to changes in oxygen tension in S. oneidensis MR-1. Biofilms of null mutants in arcA and crp were severely impacted in the stop-of-flow-induced detachment response, suggesting a role for these genes in regulation of detachment. In contrast, an DeltaetrA mutant displayed a variable detachment phenotype. From this genetic evidence we conclude that detachment is a biologically controlled process and that a rapid change in oxygen concentration is a critical factor in detachment and, consequently, in dispersal of S. oneidensis cells from biofilms. Similar mechanisms might also operate in other bacteria.

Base Sequence↗
PubMed2006

Genetic relationship and diversity in a sesame (Sesamum indicum L.) germplasm collection using amplified fragment length polymorphism (AFLP).

BACKGROUND: Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia), and to determine the association between geographical origin and genetic diversity using amplified fragment length polymorphism (AFLP). RESULTS: Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA). This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied. CONCLUSION: We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres. Future germplasm collection strategies should focus on sampling a large number of plants. Covering many diversity centres is less important because each centre represents a major part of the total diversity in sesame, Central Asia centre being the only exception. The same recommendation holds for the choice of parents for segregant populations used in breeding projects. The traditional assumption that selecting genotypes of different geographical origin will maximize the diversity available to a breeding project does not hold in sesame.

Gene Flow↗
PubMed2006

Chronic lymphocytic leukemia: current and emerging treatment approaches.

In the last decade, advances in the treatment of patients with chronic lymphocytic leukemia (CLL) have improved responses and survival outcomes dramatically. Chlorambucil had been the cornerstone of treatment for decades. Now, treatment approaches typically include a purine nucleoside analog, such as fludarabine, in combination with immunotherapy, most commonly rituximab but also alemtuzumab. Several clinical trials of patients with untreated CLL have reported overall response rates as high as 95% with chemoimmunotherapy. Combinations include fludarabine, cyclophosphamide, and rituximab (FCR); pentostatin, cyclophosphamide, and rituximab; fludarabine and rituximab; and fludarabine and alemtuzumab. FCR has demonstrated complete response rates of 70% as frontline therapy. In addition, molecular prognostic indicators, such as IgVH mutational status, ZAP-70 expression, and chromosomal mutations (trisomy 12; 17p, and 11q deletions) are proving useful in identifying patients with poor-risk disease. Furthermore, advanced technologies such as polymerase chain reaction and flow cytometry can be used to detect minimal residual disease with increasing sensitivity. Elimination of minimal residual disease has been linked to improved survival and become an important clinical goal. While overall response rates with current chemotherapy treatments are superior to those seen with single agents (eg, chlorambucil), many CLL patients still fall short of achieving a complete response. Novel agents with unique, nonoverlapping mechanisms of action (eg, oblimersen sodium, lenalidomide, HuMax-CD20, GX15-070), and other treatments are under investigation in an attempt to further improve outcomes.

Alemtuzumab↗
PubMed1991

Specificity for molecules of the major histocompatibility complex mediated by a hybrid immunoglobulin-T cell receptor.

A chimeric T-cell receptor (TCR) alpha-chain gene was produced by shuffling the immunoglobulin VDJH from a 40-140 digoxin-specific hybridoma onto alpha-chain constant region (C alpha) exons. This hybrid immunoglobulin-TCR gene was used to produce transgenic mice. Previous results indicated that this chimeric gene encoded a polypeptide that associated with endogenously encoded beta chains to form a hybrid TCR. T cells expressing this receptor could be stimulated with antibodies specific for CD3 or the 40-140 idiotype (Id40-140), and also with digoxin coupled to bovine serum albumin (digoxin-BSA). We were interested in determining whether a hybrid receptor such as this could also recognize the natural ligand of T cells, namely allelic variants of major histocompatibility complex (MHC) molecules. A T-cell hybridoma was produced that expressed a hybrid receptor with specificity for an IAk-encoded determinant, digoxin-BSA, or staphyloccocal enterotoxin B. Transfection experiments showed that the specificity for MHC determinants was dependent on both the hybrid alpha chain and a particular beta chain. These results indicate that a V beta domain combined with a VH domain can produce a receptor capable of reacting with MHC molecules, and at the same time retain specificities mediated by the beta chain and alpha chain alone. A conclusion is that the pervasive MHC specificity of the TCR is not unique to the family of TCR heterodimers, but is selected, and can be mediated by immunoglobulin domains.

Animals↗
PubMed2002

Have p53 gene mutations and protein expression a different biological significance in colorectal cancer?

p53 alterations are considered the most common genetic events in many types of neoplasms, including colorectal carcinoma (CRC). These alterations include mutations of the gene and/or overexpression of the protein. The aim of our study was to assess whether in 160 patients undergoing resective surgery for primary operable CRC there was an association between p53 mutations and protein overexpression and between these and other biological variables, such as cell DNA content (DNA-ploidy) and S-phase fraction (SPF), and the traditional clinicopathological variables. p53 mutations, identified by PCR-SSCP-sequencing analysis, were found in 68/160 patients (43%) and positive staining for p53 protein, detected with the monoclonal antibody DO-7, was present in 48% (77/160) of the cases, with agreement of 57% (91/160). In particular, a significant association was found between increased p53 expression and genetic alterations localized in the conserved regions of the gene or in the L3 DNA-binding domain and the specific type of mutation. Furthermore, both overexpression of p53 and mutations in the conserved areas of the gene were found more frequently in distal than in proximal CRCs, suggesting that they might be "biologically different diseases." Although p53 mutations in conserved areas were associated with flow cytometric variables, overexpression of p53 and mutations in its L3 domain were only related respectively to DNA-aneuploidy and high SPF. These data may reflect the complex involvement of p53 in the different pathways regulating cell-cycle progression. In conclusion, the combination of the mutational status and immunohistochemistry of p53, and flow cytometric data may provide an important insight into the biological features of CRCs.

Adult↗
PubMed2005

Local gene transfer to modulate rat corneal allograft rejection.

PURPOSE: Allograft rejection is the leading cause of corneal graft failure. CD4(+) T cells control the allograft response and represent targets for antirejection therapy. The purpose of this study was to transfer cDNA encoding a monomeric anti-CD4 antibody fragment to donor corneal endothelium, to attempt to modulate orthotopic corneal allograft rejection in the rat. METHODS: A replication-deficient adenoviral vector (AdV) encoding anti-CD4 single-chain, variable-domain antibody fragment (scFv) and enhanced green fluorescent protein (eGFP) was constructed (AdCD4GFP). AdV encoding eGFP alone (AdGFP) was used as a control. Transgenic product was detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, flow cytometry, and fluorescence microscopy. The alloinhibitory capacity of anti-rat CD4 scFv was measured in the one-way mixed lymphocyte reaction (MLR). The survival of Wistar-Furth corneas transduced with AdV either immediately or 3 days before orthotopic transplantation in Fischer 344 recipients was examined. RESULTS: ScFv and eGFP mRNAs were detected in rat corneas transduced in vitro, and active scFv secreted in corneal supernatants peaked at days 4 to 5 after transduction at 23 +/- 4 ng of protein per cornea per day. Antibody and scFv against rat CD4 blocked alloproliferation in MLR. However, transduction of corneas with AdCD4GFP ex vivo, immediately before transplantation, or in vivo, 3 days before transplantation, did not significantly prolong corneal allograft survival (P > 0.05). CONCLUSIONS: Anti-CD4 scFvs were capable of blocking allostimulation, but their local expression within the eye did not prolong corneal allograft survival, suggesting that sensitization may still occur.

Adenoviridae↗
PubMed2000

Quantitative dermatoglyphics and population structure in Northwest India.

The nature and extent of dermatoglyphic variation in northwest India is examined with the help of 28 quantitative variables-20 finger ridge counts and 8 palmar pattern ridge counts-among 12 endogamous populations. These populations represent the entire spectrum of ethnic and socioeconomic variation of the region and are presently distributed in three different states-Rajasthan, Punjab, and Himachal Pradesh. Of a total sample of 1,160 adult males, about 100 from each group were considered. Multiple discriminant analysis and R-matrix analysis were used to derive population relationships and patterns of external gene flow, respectively. Published data on genetic markers were reanalyzed to make the comparative evaluation of the patterns with reference to dermatoglyphs. Both the discriminant analysis and the F(ST) from R-matrix analysis suggest highly significant discrimination among the northwestern groups, whether one uses only 20 finger ridge count variables or all 28 variables, including the 8 palmar pattern ridge counts. The 8 palmar variables add very little to the variation explained by the 20 finger ridge count variables. F(ST) values suggest that the populations of Punjab are most homogeneous and those of Himachal Pradesh most heterogeneous. However, the levels of differentiation are similar for dermatoglyphs and genetic markers. The pattern of external gene flow as inferred through R-matrix analysis is consistent with the breeding and population structure of the groups, although genetic markers portray a relatively more realistic picture. Overall, the patterns of variation observed in dermatoglyphs and genetic markers are consistent with different dimensions of population structure; whereas dermatoglyphs conform more to the geographic pattern and less to ethnic resemblance, the reverse is true in the case of genetic markers. Am. J. Hum. Biol. 12:315-326, 2000. Copyright 2000 Wiley-Liss, Inc.

Journal Article↗
PubMed1996

Relative contribution of T and B cells to hypermutation and selection of the antibody repertoire in germinal centers of aged mice.

The immune system of aged individuals often produces antibodies that have lower affinity and are less protective than antibodies from young individuals. Recent studies in mice suggested that antibodies produced by old individuals may be encoded by distinct immunoglobulin (Ig) genes and that the somatic hypermutation process in these individuals is compromised. The present study employed Ighb scid mice reconstituted with normal lymphocytes from young (2-3-mo-old) and aged (20-25-mo-old) donors and immunized with a protein conjugate of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) to determine whether the molecular changes in antibody repertoire reflect senescence in the B cells or whether they are mediated by the aging helper T lymphocytes. The NP-reactive B cells from splenic germinal centers (GC) were recovered by microdissection of frozen tissue sections and their rearranged Ig heavy chain variable region (VH) genes of the V186.2/V3 families were sequenced. It was found that the VH gene repertoire of the GC B cells was strongly influenced by the source of the CD4+ T cells. When T cells were donated by young mice, the anti-NP response in GC was dominated by the canonical V186.2 gene, even if the responder B cells came from aged donors. However, when the mice were reconstituted with T cells from aged donors, the expression of the V186.2 gene by young B cells was diminished and the response was dominated by the C1H4 gene, another member of the V186.2/V3 family. In contrast, the somatic hypermutation process in the GC B cells followed a different pattern. The mutation frequencies in the animals that were reconstituted with both B and T cells from young donors (1/50 to 1/150 bp) were comparable to the frequencies previously reported for NP-immunized intact young/adult mice. However, when either lymphocyte subset was donated by the aged mice, the mutation frequencies declined. Thus, mice reconstituted with T cells from the aged and B cells from the young had severely compromised mutational mechanism. Likewise, the recipients of aged B and young T cells had diminished mutations even though the repertoire of their anti-NP response was dominated by the canonical V186.2 gene. It appears that the change in germine-encoded repertoire and the decrease of somatic hypermutation represent distinct mechanisms of immunosenescence and that the aging of helper T cells plays a pivotal role in both of these processes.

Aging↗
PubMed1992

T cell receptor V beta variable gene family expression in human peripheral blood lymphocytes at the mRNA and membrane protein level.

A polymerase chain reaction (PCR) was developed, using combinations of an oligonucleotide primer for a T cell receptor V beta gene family and one for the constant C beta gene segments, to assess the expression of each of 20 V beta gene families in RNA after reverse transcription into cDNA. The detection was done after agarose gel electrophoresis of PCR products and ethidium bromide staining. The positive identification of the PCR products was done by hybridization with a J beta oligonucleotide probe. For T cell lines, a signal was observed in the V beta 8 combination for Jurkat cells, V beta 5a in HSB cells, V beta 2 and V beta 12a in Molt-3 cells and V beta 2, V beta 5a and V beta 12a in Molt-4 cells. Using mixtures of RNA from different cell lines, the sensitivity of the method was in the range of 0.1-0.5%. In peripheral blood mononuclear cells from four donors, taken at three different occasions, all V beta families were detectable. The intensity of the PCR product varied between various V beta gene families. Flow cytometric analysis of blood mononuclear cells from the same donors with a restricted series of V beta gene family-specific antibodies also revealed the presence of all families. The approach to assess V beta gene family expression in heterogeneous populations opens the possibility to study T cell receptor variable gene expression in relation to physiology and pathologic processes.

Base Sequence↗
PubMed1993

Abundant mitochondrial DNA variation and world-wide population structure in humpback whales.

Hunting during the last 200 years reduced many populations of mysticete whales to near extinction. To evaluate potential genetic bottlenecks in these exploited populations, we examined mitochondrial DNA control region sequences from 90 individual humpback whales (Megaptera novaeangliae) representing six subpopulations in three ocean basins. Comparisons of relative nucleotide and nucleotype diversity reveal an abundance of genetic variation in all but one of the oceanic subpopulations. Phylogenetic reconstruction of nucleotypes and analysis of maternal gene flow show that current genetic variation is not due to postexploitation migration between oceans but is a relic of past population variability. Calibration of the rate of control region evolution across three families of whales suggests that existing humpback whale lineages are of ancient origin. Preservation of preexploitation variation in humpback whales may be attributed to their long life-span and overlapping generations and to an effective, though perhaps not timely, international prohibition against hunting.

Animals↗
PubMed1997

Cytonuclear theory for haplodiploid species and X-linked genes. I. Hardy-Weinberg dynamics and continent-island, hybrid zone models.

We develop models that describe the cytonuclear structure for either a cytoplasmic and nuclear marker in a haplodiploid species or a cytoplasmic and X-linked marker in a diploid species. Sex-specific disequilibrium statistics that summarize nonrandom cytonuclear associations in such systems are defined, and their basic Hardy-Weinberg dynamics and admixture formulae are delimited. We focus on the context of hybrid zones and develop continent-island models whereby individuals from two genetically differentiated source populations migrate into and mate within a single zone of admixture. We examine the effects of differential migration of the sexes, assortative mating by pure type females, and census time (relative to mating and migration), as well as special cases of random mating and migration subsumed under the general models. We show that pure type individuals and nonzero cytonuclear disequilibria can be maintained within a hybrid zone if there is continued migration from both source populations, and that females generally have a greater influence over these cytonuclear variables than males. The resulting theoretical framework can be used to estimate the rates of assortative mating and sex-specific gene flow in hybrid zones and other zones of admixture involving haplodiploid or sex-linked cytonuclear data.

Animals↗